The present invention relates to a method of improving the sensitivity of optical assays wherein the light signal to be analyzed emerges from the edge of an optical waveguide. The invention is particularly applicable to assays carried out using fluorescence capillary fill devices (FCFD's) in which an optical waveguide forms one plate of the device.
FCFD's and methods for their manufacture are described in detail in EP-A-171148, whilst photometric methods including assays carried out using FCFD's are described in EP-A-170376.
Assays involving FCFD's rely on angular separation, in light emerging from the optical waveguide, between fluorescence originating from molecules in sample liquid within the FCFD cavity and fluorescence orginating from molecules bound, directly or indirectly, to the waveguide. This angular separation occurs because fluorescent material in solution can only fluoresce into the waveguide at relatively large angles relative to the plane of the waveguide since the angle of any incident beam relative to this plane will be increased by refraction at the solution/waveguide interface. Such light consequently emerges from the waveguide at large angles (e.g. &gt;47.degree. relative to the axis of the waveguide). Fluorescent material bound to the surface of the waveguide, on the other hand, emits light into the waveguide at all angles, which light thereafter emerges from the waveguide over a wide range of angles relative to the axis of the waveguide. Thus, by measuring the intensity of fluorescent light emerging from the waveguide at relatively small angles to the axis of the guide (e.g. .ltoreq.45.degree. to the axis), it is possible to assess the quantity of fluorescent material bound to the surface of the waveguide since the fluorescent light output in this angular zone is substantially free from fluorescence arising from fluorescent material in solution.
As described in EP-A-171148, a convenient method of manufacturing FCFD's involves preparation of coated glass `sandwiches` from which individual FCFD's are separated by scribing and breaking. This manufacturing process permits the inexpensive bulk production of FCFD's, this being particularly desirable in view of the disposable nature of these devices.
It is difficult within the context of such a bulk production process, however, to design scribing and breaking procedures which consistently produce FCFD's in which the end of the waveguide from which emerging light is detected (hereinafter referred to as "the optical edge") is optically smooth. It will be appreciated that surface irregularities at the optical edge will give rise to some degree of light scattering and consequent mixing of the narrow angle light emission attributable only to surface-bound fluorescent material and the broader angle emission attributable to both surface-bound fluorescent material and fluorescent material in solution. This inevitably degrades the signal quality and overall performance of optical assay techniques using FCFD's.